Efficient and accurate RNA splicing is fundamentally important for proper gene expression. Group II introns provide a valuable window into the molecular mechanisms of RNA splicing and retrotransposition, and they are being harnessed as powerful tools for site-specific gene manipulation. Because of their large size and structural complexity, group II introns have provided new insights into RNA tertiary organization and folding, while also serving as a proving ground for the development of tools used in the biochemical and structural analysis of large RNA molecules. To advance all of these goals, we study the molecular structure and catalytic mechanism of group II introns. We recently solved the structure of a small, highly reactive group II intron in the spliced product state. This work revealed an elaborate architectural frame that surrounds a conserved active-site motif for binding catalytic metal ions. Our continuing projects will build on these findings by revealing the multiple conformational states that are adopted during the different stages of splicing and by revealing the structural features of group II intron-maturase complexes. At each phase of the project, structural information will be used to guide functional studies on molecular mechanisms of splicing and retrotransposition. There are three complementary aims of this project. In AIM 1, we will crystallographically characterize the conformational states of the Oceanobacillus iheyensis group IIC intron, enabling us to visualize its function as a molecular machine during splicing. We will also characterize the folding pathway of this intron and identify the molecular determinants for transition- state stabilization during catalysis. In AIM 2, we will structurally characterize the more elaborate group II intron subtype that maintains high levels of sequence specificity and which contains novel architectural motifs. In AIM 3, we will solve the structure of a group II intron-maturase RNP, which is the natural form of the intron that typically functions during intron mobility and splicing. Using a combination of x-ray crystallography, solution biochemical structure mapping and mechanistic enzymology, we will reveal the respective contributions of RNA and protein to the processes of group II intron splicing and mobility, providing information that is essential for our understanding of molecular evolution and for the development of group II introns as tools and potential therapeutics.